Journal of Comparative and Physiological Psycholoa 1975, Vol. 89, No. 9, 1061-1069
Telson Reflex Habituation in Limulus polyphemus Robert Lahue, Larry Kokkinidis, and William Corning University of Waterloo, Waterloo, Ontario, Canada Muscle correlates of reflex telson movement were recorded in intact Limulus (horseshoe crab) preparations with chronically implanted microelectrodes. Muscle activity habituated during repetitive tactile stimulation of the gills with puffs of air. Dishabituation was also observed, as were inverse relationships between the frequency and intensity of stimulation and the rate of response decrement. These findings closely paralleled those obtained during central recordings in acute dissected preparations, as did the demonstration under certain stimulation conditions of an initial incremental phase of responsivity. Although initial levels of responsivity recovered after 1 hr without stimulation, potentiation of habituation was observable after 2 hr. Both of these times greatly exceeded those previously observed in acute preparations. No generalization to a spatial displacement of the stimulus was obtained, although a visually elicited telson reflex had been shown to demonstrate a cross-optic generalization. Aspects of the comparative strategy and the adaptive value of telson reflex habituation in Limulus were briefly considered.
of a subject. Too often in simple-system research, a preparation is selected that permits the application of some technique because of its elegance rather than because it offers some behaviorally relevant characteristic (Abraham, Palka, Peeke, & Willows, 1972). Additionally, demonstrations of plasticity at the neuronal level do not always parallel the behavior of the intact animal (e.g., Krasne, 1973). Some compromise is needed with respect to ease of technical application and behavioral suitability. With respect to habituation, Limulus represents a potentially good candidate for a multilevel-polythetic strategy. The large central nervous system permits easy dissection and location of specific ganglia; microelectrode, perfusion, and lesion techniques are readily applied to any portion of the nervous system in acute preparations (Corning, Lahue, & Von Burg, 1971; Von Burg & Corning, 1970, 1971); and methods for chronic monitoring in relatively unrestrained animals have also been developed (Adolph, 1971; Corning, Feinstein, & Haight, 1965). Evidence for habituation has been estabThis research was supported in part by Grant lished at the neuronal level (Lahue & CornAPA0351 from the National Research Council of ing, 1971), and the neurophysiological and Canada to W. Corning. Requests for reprints should be sent to Robert biochemical mechanisms of this form of plasLahue, Psychology Department, University of ticity are beginning to be understood (LaWaterloo, Waterloo, Ontario, Canada N2L 3G1. hue, 1974; Lahue & Corning, 1973). The 1061
The "simple system" strategy, which relies upon surgery or phylogeny to provide preparations of limited complexity, is resulting in considerable information with respect to learning mechanisms (Corning, Dyal, & Willows, 1973a, 1973b, 1974; Kandel & Spencer, 1968). However, as potential substrates of behavior are uncovered, it is becoming increasingly clear that a more conservative approach toward comparative generalizations is required, since many similar processes observed across species are found to be subserved by dissimilar mechanisms (Corning & Lahue, 1972; Dyal & Corning, 1973). The establishment of comparative similarities and dissimilarities requires a "polythetic" strategy, which compares and classifies systems or processes according to multiple criteria and at many levels of biological organization (Corning, Dyal, & Lahue, in press; Lahue & Corning, 1974; Sneath & Sokal, 1973). If multiple criteria and levels are required for sounder comparative analyses, then a critical consideration involves the selection
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R. LAHUE, L. KOKKINIDIS, AND W. CORNING
basic paradigm in these neuronal investiga- gered by a square-wave stimulator. Each puff was tions was to use a puff of air or a drop of 250 msec in duration. A brief squirt of saline from a syringe was used as a dishabituation stimulus. water applied to the gills as a stimulus and Telson muscle and heart recordings were achieved to record the responses of units in the dorsal differentially through capacity-coupled, highnerves of the abdominal ganglia. To explore impedance amplifiers (Grass P511), and amplified the usefulness of Limulus at complex as well output was displayed on a Beckman Type R dynagraph. Muscle activity was also integrated and as simple-system levels, this basic paradigm polygraphically displayed to facilitate data was applied to intact preparations. To assess analysis. (See Figure 2 for an example of the data the true comparability between the acute display.) After surgery the animals were returned to and intact preparations and between Limulus and other species, the following multiple their home tank, fed on the following day, and alto recover for at least 10 days before use in habituation criteria, as outlined by Thomp- lowed experiments. For all experiments, the animals son and Spencer (1966), were examined: were fastened to a board by nails driven through habituation, spontaneous recovery, poten- the edge of the carapace and/or by clamps. The tiation of habituation, frequency effects, in- telson extended over the edge of the board and was free to move in all directions. A hole in the board tensity effects, stimulus generalization, and exposed the gills to stimulation. dishabituation. In general, after an animal was set up for exGENERAL METHOD Specimens of Limulus, measuring 6-8 in. (15-20 cm) across the cephalothorax, were obtained from Marine Biological Supplies, Woods Hole, Massachusetts. The animals were kept in tanks of artificial sea water (Instant Ocean) at a specific gravity of 1.024, pH of 7.8-8.3, and temperature of 1518 °C. The water was constantly filtered and aerated. The animals were force fed minced clams about twice a month. Electrodes for chronic monitoring of heart rate and telson muscle activity were implanted. The beveled end of a small Plexiglas cannula was inserted into a .5-cm hole drilled through the carapace just above a telson extensor muscle. An insulated tungsten microelectrode was passed through a .01-in (.25-mm) hole in the center of the cannula and positioned with a mieromanipulator to achieve the best recording of muscle activity. All implants were checked to determine that only spontaneous movement of the telson produced electrical activity. If passive manipulation of the telson by the experimenter produced any mechanical artifacts, the preparation was not used. Two stainless steel screws were inserted through the carapace just posterior to the hinge for heart recordings. The heart electrodes, muscle electrode, and other stainless steel screws used as differential and ground electrodes were all fixed to the animal and buried with dental acrylic after lead wires had been soldered to them. Stimuli presented to specific points on the gillbook surface were puffs of air. The air was delivered through a pipette with a 1-mm tip diameter, which was positioned about 5-7 mm from the animal. Air pressure was provided by an air pump or a compressed-air cylinder, and the air was passed through a gas-washing bottle. Unless otherwise noted, the system was pressurized at 4 Ibs. (.28 kg/cm 2 ). Stimulus frequency and duration were controlled by an Asco two-way valve trig-
perimentation, 30 min were allowed to elapse before stimulation in order to establish a base rate for spontaneous activity. Calculation of the area under the curve of integrated muscle activity provided a quantitative estimate of response amplitude and duration. Since many factors, including electrode positioning and animal size, affected the absolute size of the response as recorded on the chart paper, a normalization procedure was used to allow comparisons between animals. The amplitude of the response on the first trial was divided into that obtained on each successive trial and then multiplied by 100. The data, therefore, are in terms of percent responses, with the response on the first trial considered as 100%. Because of a rather large heterogeneity of variance, all statistical tests used were nonparametric as described by Siegel (1956).
EXPERIMENT 1 The purpose of Experiment 1 was to examine the general time course of habituation and spontaneous recovery. In addition, the relationship between recovery and potentiation of habituation was investigated. A dishabituation stimulus was used to determine whether muscle fatigue was responsible for any response decrements observed. Method Each of nine animals was habituated, using a series of 20 air puffs with a 30-sec intertrial interval (ITI). This was followed by a squirt of water applied to the habituation site. The same sequence was repeated during retests given 1, 2, or 4 hr after the last acquisition stimulus. Each animal was tested on all three intervals with a minimum of 48 hr between successive tests. The retest interval was randomly presented. Additionally, the effects of repeated testing at 24-hr intervals were investi-
HABITTJATION IN HORSESHOE CRAB gated; subjects were given the series of 20 air puffs for four successive days.
Results and -Discussion The total response magnitude summed over 20 trials did not vary significantly on four successive days, Kruskal-Wallis analysis of variance, H(3) = 1.5, p > .05. Thus, no retention of habituation was observed after a 24-hr spontaneous recovery period. Furthermore, since repeated testing does not affect habituation with intervals of 24 hr, animals could be tested more than once in other experiments. A minimum 48-hr interval always intervened between successive tests on the same animal in all other conditions. No significant differences were observed during acquisition among the groups in the shorter retention tests, H(2) = .0, p > .05. Differences were observed during retest after various rest periods, H(2) = 12.12, p < .01. The 1- and 2-hr groups did not significantly differ from one another (Mann-Whitney U test, U = 36, p > .05), but both groups did differ from the 4-hr retest group (U = 1 2 , p < .002 for both comparisons). The 1- and 2-hr retests differed from initial acquisition (Wilcoxon sign test, T = 4, p < .05 and T = 2, p < .02, respectively), but there was no significant difference after a 4-hr rest period (T = 12, p > .05). The data for the three retention tests arc presented in Figure 1. As can be noted in Figure 1, the responses to the first stimulus on acquisition and on each of the retests are essentially the same (H = 1.61, p > .05). Thus, if the criterion for spontaneous recovery is a return to initial levels of responsivity, the process is complete within an hour. However, potentiation of habituation, as indicated by a more rapid response decrement on retest when compared with acquisition, is still apparent after a 2-hr rest period as indicated above. The response to the dishabituation stimulus (water jet) for all groups after both acquisition and retest was significantly larger than the response to the last air puff (T < 4, p < .05 in all cases) but did not differ from the response to the first stimulus (T > 12, p > .05 in all cases). A marked cardioinhibition was always ob-
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served after the first stimulus of a series. However, subsequent stimulus effects upon electrocardiograms (EGG) were extremely transient and not always well defined (see Figure 2). Because of the transient nature of the EGG responses in the present experimental conditions, they were not analyzed. Several of the Thompson and Spencer (1966) criteria have, then, been fulfilled. A response decrement did occur with repeated stimulation. After as little as 1 hr, spontaneous recovery returned responsivity to initial levels. However, the rate of decrement after as much as 2 hr of spontaneous recovery was greater than that observed during acquisition, indicating potentiation of habituation. Dorsal nerve responsivity in surgical preparations recovers in as little as 12 min, and potentiation effects are not seen when intervals of 30 min separate series of stimuli (Lahue & Corning, 1971). A similar disparity between retention times in Aplysia preparations has also been reported (Carew, Pinsker, & Kandel, 1972). Responsiveness to a novel stimulus following habituation demonstrates that effector fatigue is not the basis for the response decrement, and sensory receptor fatigue had been ruled out in previous investigations of acute preparations (Lahue, 1974; Lahue & Corning, 1971).
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FIGURE 1. Percent change in telson muscle activity during the first 300 sec (11 trials) of an initial test and a retest after three different spontaneous recovery periods. (The initial test data for each of the 3 test-retest pairings have been combined.)
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EXPERIMENT 2 Having established that habituation occurs to the air puff and that some of the primary habituation criteria could be met, we examined further criteria in Experiment 2. The effect of the frequency of stimulation upon habituation and the influence of dishabituation stimuli upon responsivity to subsequent stimuli were investigated. Method A total of 11 animals was tested at four different stimulus frequencies. At least 48 hr intervened between successive tests on a single animal, and the order of presentation of the different frequency conditions was randomized. A total of 40 air-puff stimuli were presented with ITIs of 60 sec (n = 9), 30 sec (n = 9), 15 sec (n = 11), and 5 sec (n = 11). Between Trials 20 and 21 a jet of water (dishabituation stimulus) was directed at the stimulation site.
Results and Discussion The data presented in Figure 3 are plotted with respect to time on the abscissa.
Analyses of variance of the differences among the groups were performed on the data obtained 60 and 120 sec after the beginning of stimulation as well as immediately after the dishabituation stimulus, and 60 and 120 sec thereafter. Comparisons were also made between the 30- and 60-sec groups 540 sec after both the first stimulus and the dishabituation stimulus. Three analyses, then, were performed on the data obtained during the first 20 trials. After 60 sec of stimulation, there were significant differences between all groups (H = 24.53, p < .001; U < 24.5, p < .02 for six comparisons). After 120 sec, similar results were obtained (H = 20.77, p < .001; U < 24.5, p < .02 for six comparisons). At 540 sec, the 30- and 60-sec groups differed significantly (U = 69, p < .02). There were no differences among the groups in the magnitude of the response to the dishabituation stimulus (H = 3.97, p > .05). However, there were significant differences in responsivity to air puffs immediately following the
HABITUATION IN HORSESHOE CRAB
presentation of the jet of water (H = 21.15, p < .001; U < 22.0, p < .02 for six comparisons). There were no significant differences between the 5- and 15-sec groups 60 and 120 sec after the dishabituation stimulus. However, all other comparisons among the four groups were significant at these two points in time (H > 20.91; p < .001; U < 24.5, p < .02 for all comparisons). The 30- and 60-sec groups also differed from one another 540 sec after the dishabituation stimulus (U = 64, p < .004). Total response magnitudes (the sum of the responses at time intervals 0, 60, 120, and 540 sec) exhibited significant differences when comparisons were made with the 20 trials before the dishabituation stimulus and those after the dishabituation stimulus for each of the four groups (T < 6, p < .02 for all four comparisons). The amplitudes of the responses to the first air puff following the dishabituation stimulus were significantly larger than those elicited by the previous air-puff stimulus for all four groups (T < 5, p < .05 in each case). The usual relationship between frequency of stimulation and rate of habituation was obtained: Higher frequencies produced more rapid decrement. The rate of habituation at the different frequencies rather closely paralleled the decrement seen in dorsal nerve recordings, although less habituation was observed in the latter with a 60-sec ITI. The possibility of demonstrating response sensitization at higher frequencies of stimulation (e.g., 1-sec ITI), as was observed in dorsal nerve recordings (Lahue & Corning, 1973),
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was precluded by the duration of the telson response, which approached 5 sec. True dishabituation was demonstrated in that the response to air on the 21st trial exceeded that prior to the dishabituation stimulus. The recovery was not complete as the posthabituation response was not so large as that elicited by the first stimulation. The degree of recovery following the dishabituation stimulus and the subsequent rate of habituation were both related to the frequency of stimulation. EXPERIMENT 3 Two further habituation criteria were examined in this experiment. The effects of stimulus intensity upon habituation to airpuff stimuli were tested in the first part, and stimulus generalization to a spatial displacement of the stimulus site was investigated in the second part. Method A total of 11 animals was used in this experiment. All subjects were tested under high-stimulus intensity conditions; nine of these were also tested with a low-intensity stimulus, and the order of presentation of the high- and low-intensity conditions was randomized. At least 1 wk intervened between the two tests for a given animal. The intensities of the stimuli used in these experiments were controlled by altering the pressure in the air reservoir.2 The pressures used were either 1 Ib.2 (.07 kg/cm ; low intensity) or 8 Ib. (.56 kg/cm ; high intensity). A total of 20 air-puff stimuli was presented, with a 30-sec ITI followed by a dishabituation test with a jet of water. Nine of these animals were subsequently presented (a minimum of 6 days later) with two series of 20 air-
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puff .stimuli (30-seo ITI) at the normal intensity (4 lb.; .28 kg/em 2 ). After the initial 20 trials the stimulus was immediately moved to another site, approximately homo topic, on the contralateral side of the animal. An additional 20 stimuli were directed at this site. The responses quantified during each series were normalized separately as described above. The response on the first trial in each series was used as the 100% response for the calculation of subsequent trials in the same series. This was necessary to allow comparison between the Jwo series of responses because the initial responses elicited by stimulation of different sites often varied in amplitude.
Results and Discussion As seen in Figure 4, the total responsivity during high-intensity stimulation exceeds that during low-intensity stimulation (U = 8, p < .002). The high-intensity stimulus diminishes the rate of habituation. This is due, in part, to an initial period of incremental responding and suggests a dualprocess possibility (Groves & Thompson, 1970). State or sensitizing influences upon the output are thought to be enhanced by strong stimuli and to compete with the habituation process. Analysis of the response to the water jet (dishabituation stimulus) in the two groups shows that the high-intensity group is more responsive (U = 23, p < .05). This suggests that state has been activated and some residual influence upon responsivity is seen even after habituation. Previous experi-
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'»•*• .05). Although Thompson and Spencer (1966) considered this an important criterion for habituation, Hinde (1970) suggested that a certain amount of stimulus specificity is also characteristic of this process. Not all invertebrates demonstrate generalization, and some exhibit only one type (e.g., generalization only to stimuli of different modalities presented to the same site or only to stimuli of the same modality presented to different sites; see Corning & Lahue, 1972; Dyal & Corning, 1973). GENERAL DISCUSSION The data obtained in the present investigations demonstrate that most of the habituation criteria that are outlined by Thompson and Spencer (1966) can be met in a relatively intact, chronic Limulus preparation. With the previously obtained evidence that these criteria can be generally fulfilled in acute and surgically reduced preparations (Lahue, 1974), polythetic comparisons are possible with both intact and simplified preparations of other species. Although few investigators gathered information on all nine criteria, there are a sufficient number of examples to make some tentative phyletic comparisons. Thus, for example, in acute surgical preparations of the locust Schistocerca gregaria (Horn & Rowell, 1968; Rowell, 1971) most of the criteria were fulfilled (intensity effects were not tested). Very little spatial generalization of habituation was observed. The time course of spontaneous recovery was rather long compared with that of surgically limited Limulus preparations, al-
HABITUATION IN HORSESHOE CRAB
though similar to that observed in intact preparations. Krasne (1969, 1973; Krasne & Woodsmall, 1969) also demonstrated the same habituation criteria, including generalization but excluding dishabituation, in crayfish abdominal ganglia. Generally, differences in the time course of habituation are seen in intact versus acute preparations, and the process observed in acute preparations does not correspond fully with the properties of habituation shown in intact animals. The Thompson and Spencer criteria should not be considered as rigid "laws" around which all comparative statements concerning habituation persist or fall. They represent guidelines which will probably shift and expand as additional investigations explore more species with respect to multiple criteria. Some of the problems concerning these criteria have already been discussed (Hinde, 1970; Lahue, 1974), and it may be that certain aspects of the habituation process will become less critical; for example, in the present study there was no generalization of habituation when a homotopic site was stimulated. A specificity in the decremental process is thus obtained in the abdominal ganglia, but in Limulus visual-motor reflexes, cross-optic generalization had been observed (Coming & Von Burg, 1968). The generalization process may become less important as a criterion that is used to classify the general process of habituation amd more of a "subset marker" (Dyal & Corning, 1973). Those criteria that have greater phylogenetic ubiquity (decrement, spontaneous recovery, frequency effects) may be considered critical for the general category. Accordingly, in Limulus two types of habituation are possible: a decrement with stimulus generalization in visual-motor reflexes and one without generalization in tactile reflexes mediated by the abdominal ganglia. The usefulness of this criterion and others will be expanded when they prove sensitive to phyletic differences and to structural and physiological variations. To summarize, comparisons between acute and chronic studies show a number of similarities. The rates of habituation in both cases were similar over a range of stimulus frequencies. Although the higher frequencies
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of stimulation, which were used to reveal the dual process of habituation in prior studies (Lahue & Corning, 1973), could not be utilized, a higher stimulus intensity did produce the incremental phase. Responsiveness to novel stimuli after habituation was also similar in both cases. The reintroduction of normal stimulation after the novel stimulus resulted in higher responses for a few trials. An interaction of the dishabituation effect with stimulus frequency was found, although this has not been previously reported in the literature. Stimulus generalization was not demonstrable in the intact animal; previous research with acute preparations did not systematically examine whether generalization was obtainable in surgically reduced systems. Discovery of a dual process (increment and decrement) during repetitive stimulation of isolated ganglia added to the complexity of habituation in Limulus (Lahue & Corning, 1973). A dual-process theory, as originally put forth by Groves and Thompson (1970), seems likely to apply to a wide variety of invertebrate and vertebrate preparations. The evidence obtained with the present preparation indicates that in Limulus the two processes are observed in the intact animal and are not artifacts of surgical procedures. With more intense stimulation the incremental phase is apparent. The two-process theory is most likely incomplete. In acute preparations evidence has been obtained that a third process—sensory modulation— interacts with the incremental and decremental processes (Lahue & Corning, 1973). Since the overall characteristics of habituation are similar in both acute and intact cases, it can be hoped that in surgically reduced preparations the elucidation of processes and mechanisms will have relevance to intact animals. The striking difference between the two preparations was in the persistence or retention of the habituation effect. As with other simple-system preparations, the performance of the intact animal is superior (e.g., Krasne, 1973). One obvious possibility is that the surgery results in metabolic degeneration. It is also possible that in the acute preparation, the trauma of surgery in-
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R. LAHUE, L. KOKKINIDIS, AND W. CORNING
creases central state and thereby counteracts the acquisition and retention phases. A third possibility is that because of metabolic degeneration, deliberate surgical isolation, or undetermined damage, the system is reduced structurally, i.e., it is less complex. From direct examination of the relationship between system complexity and habituation, it is known that the more intact system performs best (Corning & Lahue, 1972; Lahuc & Corning, 1971). The failure to find significant and useful effects of stimulation on heart rate was unexpected, since prior studies had shown light and tactile effects (Corning & Von Burg, 1968; Von Burg & Corning, 1970). However, in these previous studies stimulation was longer and more intense and was not given repetitively with short ITIs for the purpose of examining habituation. The results of the present study suggest that with the particular stimulus characteristics that were used, the heart was not a good indicant of stimulation effects because habituation was very rapid and/or the stimuli were weak with respect to this system. The telson muscles, however, represent a sufficiently sensitive Finally, a question should be raised concerning the adaptive significance of habituation to tactile input. The locus of stimulation used in the present investigation was the surface of the gills. Since the animal is a bottom dweller and not prone to swim as an adult, normal movements will result in consistent stimulation of the gills. Additionally, normal respiratory activities of the gills result in tactile input as they fold over each other, an activity that produces regular stimulation. Both sources of input could conceivably be disruptive to normal biological and behavioral activities if habituation were not possible—the heart rate might be continually interrupted and the telson would be frequently jerking upward as the system responded to each burst of tactile input. REFERENCES Abraham, F., Palka, J., Peeke, H. V. S., & Willows, A. O. D. Model neural systems and strategies for the neurobiology of learning. Behavioral Biology, 1972, 7,1-24. Adolph, A. R. Recording of optic nerve spikes
underwater from freely-moving horseshoe crab. Vision Research, 1971,11, 979-983. Carew, T. J., Pinsker, H. M., & Kandel, E. R. Long-term habituation of a defensive withdrawal reflex in Aplysia. Science, 1972, 175, 451-454. Corning, W. C., Dyal, J. A., & Lahue, R. Intelligence: An invertebrate perspective. In B. Masterton, M. E. Bitterman, W. Hodos, & H. Jerison (Eds.), Evolution of the nervous system and behavior. Boston: Winston, in press. Corning, W. C., Dyal, J. A., & Willows, A. O. D. (Eds.)- Invertebrate learning (Vol. 1). New York: Plenum Press, 1973. (a) Corning, W. C., Dyal, J. A., & Willows, A. O. D. (Eds.). Invertebrate learning (Vol. 2). New York: Plenum Press, 1973. (b) Corning, W. C., Dyal, J. A., & Willows, A. O. D. (Eds.). Invertebrate learning (Vol. 3). New York: Plenum Press, 1974. Corning, W. C., Feinstein, D. A., & Haight, J. H. Arthropod preparation for behavioral, electrophysiological and biochemical studies. Science, 1965, 148, 394-395. Corning, W. C., & Lahue, R. Invertebrate strategies in comparative learning studies. American Zoologist, 1972, IS, 455-469. Corning, W. C., Lahue, R., & Von Burg, R. Supra esophageal ganglia influences on Limulus heart rhythm: Confirmatory evidence. Canadian Journal of Physiology and Pharmacology, 1971, 49, 387-393. Corning, W. C., & Von Burg, R. Behavioral and neurophysiological investigations of Limulus polyphemus. In J. Salanki (Ed.), Neurobiology of invertebrates. New York: Plenum Press, 1968. Dyal, J. A., & Corning, W. C. Invertebrate learning and behavioral taxonomies. In W. C. Corning, J. A. Dyal, & A. O. D. Willows (Eds.), Invertebrate learning (Vol. 1). New York: Plenum Press, 1973. Groves, P. M., & Thompson, R. F. Habituation: A dual-process theory. Psychological Review, 1970, 77, 419-450. Hinde, R. A. Behavioral habituation. In G. Horn & R. A. Hinde (Eds.), Short-term changes in neural activity and behavior. Cambridge, England: Cambridge University Press, 1970. Horn, G., & Rowell, C. H. F. Medium and longterm changes in the behavior of visual neurones in the tritocerebrum of locusts. Journal of Experimental Biology, 1968, 49, 143-169. Kandel, E. R., & Spencer, W. A. Cellular neurophysiological approaches in the study of learning. Physiological Reviews, 1968, 4$, 65-134. Krasne, F. B. Excitation and habituation of the crayfish escape reflex: The depolarizing response in lateral giant fibres of the isolated abdomen. Journal of Experimental Biology, 1969, BO, 29-46. Krasne, F. B. Learning in Crustacea. In W. C. Corning, J. A. Dyal, & A. O. D. Willows (Eds.), Invertebrate learning (Vol. 2). New York: Plenum Press, 1973.
HABITUATION IN HORSESHOE CRAB Krasne, F. B., & Woodsmall, K. S. Waning of the crayfish escape response as a result of repeated stimulation. Animal Behaviour, 1969,77,416-424. Lahue, R. Habitualion characteristics and mechanisms in the abdominal ganglia of Limulus polyphemus. Unpublished doctoral dissertation, University of Waterloo, 1974. Lahue, R., & Corning, W. C. Habituation in Limulus abdominal ganglia. Biological Bulletin, 1971, 140, 427-439. Lahue, R., & Corning, W. C. Incremental and decremental processes in Limulus ganglia: Stimulus frequency and. ganglion organization influences. Behavioral Biology, 1973,8,637-653. Lahue, R., & Corning, W. C. Synthesis: A comparative look at vertebrates. In W. C. Corning, J. A. Dyal, & A. O. D. Willows (Eds.), Invertebrate learning (Vol. 3). New York: Plenum Press, 1974. Rowell, C. H. F. Antennal cleaning, arousal and
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visual interneurone responsiveness in a locust. Journal of Experimental Biology, 1971, 55, 749761. Siegel, S. Nonparametric statistics. New York: McGraw-Hill, 1956. Sneath, P., & Sokal, R. Principles of numerical taxonomy. San Francisco: Freeman, 1973. Thompson, R. P., & Spencer, W. A. Habituation: A model phenomenon for the study of the neuronal substrates of behavior. Psychological Review, 1966, 178, 16-43. Von Burg, R., & Corning, W. C. Cardioregulatory properties of Limulus abdominal ganglia. Canadian Journal of Physiology and Pharmacology, 1970, 48, 333-341. Von Burg, R., & Corning, W. C. The effects of drugs on Limulus cardioregulators. Canadian Journal of Physiology and Pharmacology, 1971, 49, 1044-1048. (Received November 21, 1974)
Telson Reflex Habituation in Limulus polyphemus Robert Lahue, Larry Kokkinidis, and William Corning University of Waterloo, Waterloo, Ontario, Canada Muscle correlates of reflex telson movement were recorded in intact Limulus (horseshoe crab) preparations with chronically implanted microelectrodes. Muscle activity habituated during repetitive tactile stimulation of the gills with puffs of air. Dishabituation was also observed, as were inverse relationships between the frequency and intensity of stimulation and the rate of response decrement. These findings closely paralleled those obtained during central recordings in acute dissected preparations, as did the demonstration under certain stimulation conditions of an initial incremental phase of responsivity. Although initial levels of responsivity recovered after 1 hr without stimulation, potentiation of habituation was observable after 2 hr. Both of these times greatly exceeded those previously observed in acute preparations. No generalization to a spatial displacement of the stimulus was obtained, although a visually elicited telson reflex had been shown to demonstrate a cross-optic generalization. Aspects of the comparative strategy and the adaptive value of telson reflex habituation in Limulus were briefly considered.
of a subject. Too often in simple-system research, a preparation is selected that permits the application of some technique because of its elegance rather than because it offers some behaviorally relevant characteristic (Abraham, Palka, Peeke, & Willows, 1972). Additionally, demonstrations of plasticity at the neuronal level do not always parallel the behavior of the intact animal (e.g., Krasne, 1973). Some compromise is needed with respect to ease of technical application and behavioral suitability. With respect to habituation, Limulus represents a potentially good candidate for a multilevel-polythetic strategy. The large central nervous system permits easy dissection and location of specific ganglia; microelectrode, perfusion, and lesion techniques are readily applied to any portion of the nervous system in acute preparations (Corning, Lahue, & Von Burg, 1971; Von Burg & Corning, 1970, 1971); and methods for chronic monitoring in relatively unrestrained animals have also been developed (Adolph, 1971; Corning, Feinstein, & Haight, 1965). Evidence for habituation has been estabThis research was supported in part by Grant lished at the neuronal level (Lahue & CornAPA0351 from the National Research Council of ing, 1971), and the neurophysiological and Canada to W. Corning. Requests for reprints should be sent to Robert biochemical mechanisms of this form of plasLahue, Psychology Department, University of ticity are beginning to be understood (LaWaterloo, Waterloo, Ontario, Canada N2L 3G1. hue, 1974; Lahue & Corning, 1973). The 1061
The "simple system" strategy, which relies upon surgery or phylogeny to provide preparations of limited complexity, is resulting in considerable information with respect to learning mechanisms (Corning, Dyal, & Willows, 1973a, 1973b, 1974; Kandel & Spencer, 1968). However, as potential substrates of behavior are uncovered, it is becoming increasingly clear that a more conservative approach toward comparative generalizations is required, since many similar processes observed across species are found to be subserved by dissimilar mechanisms (Corning & Lahue, 1972; Dyal & Corning, 1973). The establishment of comparative similarities and dissimilarities requires a "polythetic" strategy, which compares and classifies systems or processes according to multiple criteria and at many levels of biological organization (Corning, Dyal, & Lahue, in press; Lahue & Corning, 1974; Sneath & Sokal, 1973). If multiple criteria and levels are required for sounder comparative analyses, then a critical consideration involves the selection
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R. LAHUE, L. KOKKINIDIS, AND W. CORNING
basic paradigm in these neuronal investiga- gered by a square-wave stimulator. Each puff was tions was to use a puff of air or a drop of 250 msec in duration. A brief squirt of saline from a syringe was used as a dishabituation stimulus. water applied to the gills as a stimulus and Telson muscle and heart recordings were achieved to record the responses of units in the dorsal differentially through capacity-coupled, highnerves of the abdominal ganglia. To explore impedance amplifiers (Grass P511), and amplified the usefulness of Limulus at complex as well output was displayed on a Beckman Type R dynagraph. Muscle activity was also integrated and as simple-system levels, this basic paradigm polygraphically displayed to facilitate data was applied to intact preparations. To assess analysis. (See Figure 2 for an example of the data the true comparability between the acute display.) After surgery the animals were returned to and intact preparations and between Limulus and other species, the following multiple their home tank, fed on the following day, and alto recover for at least 10 days before use in habituation criteria, as outlined by Thomp- lowed experiments. For all experiments, the animals son and Spencer (1966), were examined: were fastened to a board by nails driven through habituation, spontaneous recovery, poten- the edge of the carapace and/or by clamps. The tiation of habituation, frequency effects, in- telson extended over the edge of the board and was free to move in all directions. A hole in the board tensity effects, stimulus generalization, and exposed the gills to stimulation. dishabituation. In general, after an animal was set up for exGENERAL METHOD Specimens of Limulus, measuring 6-8 in. (15-20 cm) across the cephalothorax, were obtained from Marine Biological Supplies, Woods Hole, Massachusetts. The animals were kept in tanks of artificial sea water (Instant Ocean) at a specific gravity of 1.024, pH of 7.8-8.3, and temperature of 1518 °C. The water was constantly filtered and aerated. The animals were force fed minced clams about twice a month. Electrodes for chronic monitoring of heart rate and telson muscle activity were implanted. The beveled end of a small Plexiglas cannula was inserted into a .5-cm hole drilled through the carapace just above a telson extensor muscle. An insulated tungsten microelectrode was passed through a .01-in (.25-mm) hole in the center of the cannula and positioned with a mieromanipulator to achieve the best recording of muscle activity. All implants were checked to determine that only spontaneous movement of the telson produced electrical activity. If passive manipulation of the telson by the experimenter produced any mechanical artifacts, the preparation was not used. Two stainless steel screws were inserted through the carapace just posterior to the hinge for heart recordings. The heart electrodes, muscle electrode, and other stainless steel screws used as differential and ground electrodes were all fixed to the animal and buried with dental acrylic after lead wires had been soldered to them. Stimuli presented to specific points on the gillbook surface were puffs of air. The air was delivered through a pipette with a 1-mm tip diameter, which was positioned about 5-7 mm from the animal. Air pressure was provided by an air pump or a compressed-air cylinder, and the air was passed through a gas-washing bottle. Unless otherwise noted, the system was pressurized at 4 Ibs. (.28 kg/cm 2 ). Stimulus frequency and duration were controlled by an Asco two-way valve trig-
perimentation, 30 min were allowed to elapse before stimulation in order to establish a base rate for spontaneous activity. Calculation of the area under the curve of integrated muscle activity provided a quantitative estimate of response amplitude and duration. Since many factors, including electrode positioning and animal size, affected the absolute size of the response as recorded on the chart paper, a normalization procedure was used to allow comparisons between animals. The amplitude of the response on the first trial was divided into that obtained on each successive trial and then multiplied by 100. The data, therefore, are in terms of percent responses, with the response on the first trial considered as 100%. Because of a rather large heterogeneity of variance, all statistical tests used were nonparametric as described by Siegel (1956).
EXPERIMENT 1 The purpose of Experiment 1 was to examine the general time course of habituation and spontaneous recovery. In addition, the relationship between recovery and potentiation of habituation was investigated. A dishabituation stimulus was used to determine whether muscle fatigue was responsible for any response decrements observed. Method Each of nine animals was habituated, using a series of 20 air puffs with a 30-sec intertrial interval (ITI). This was followed by a squirt of water applied to the habituation site. The same sequence was repeated during retests given 1, 2, or 4 hr after the last acquisition stimulus. Each animal was tested on all three intervals with a minimum of 48 hr between successive tests. The retest interval was randomly presented. Additionally, the effects of repeated testing at 24-hr intervals were investi-
HABITTJATION IN HORSESHOE CRAB gated; subjects were given the series of 20 air puffs for four successive days.
Results and -Discussion The total response magnitude summed over 20 trials did not vary significantly on four successive days, Kruskal-Wallis analysis of variance, H(3) = 1.5, p > .05. Thus, no retention of habituation was observed after a 24-hr spontaneous recovery period. Furthermore, since repeated testing does not affect habituation with intervals of 24 hr, animals could be tested more than once in other experiments. A minimum 48-hr interval always intervened between successive tests on the same animal in all other conditions. No significant differences were observed during acquisition among the groups in the shorter retention tests, H(2) = .0, p > .05. Differences were observed during retest after various rest periods, H(2) = 12.12, p < .01. The 1- and 2-hr groups did not significantly differ from one another (Mann-Whitney U test, U = 36, p > .05), but both groups did differ from the 4-hr retest group (U = 1 2 , p < .002 for both comparisons). The 1- and 2-hr retests differed from initial acquisition (Wilcoxon sign test, T = 4, p < .05 and T = 2, p < .02, respectively), but there was no significant difference after a 4-hr rest period (T = 12, p > .05). The data for the three retention tests arc presented in Figure 1. As can be noted in Figure 1, the responses to the first stimulus on acquisition and on each of the retests are essentially the same (H = 1.61, p > .05). Thus, if the criterion for spontaneous recovery is a return to initial levels of responsivity, the process is complete within an hour. However, potentiation of habituation, as indicated by a more rapid response decrement on retest when compared with acquisition, is still apparent after a 2-hr rest period as indicated above. The response to the dishabituation stimulus (water jet) for all groups after both acquisition and retest was significantly larger than the response to the last air puff (T < 4, p < .05 in all cases) but did not differ from the response to the first stimulus (T > 12, p > .05 in all cases). A marked cardioinhibition was always ob-
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served after the first stimulus of a series. However, subsequent stimulus effects upon electrocardiograms (EGG) were extremely transient and not always well defined (see Figure 2). Because of the transient nature of the EGG responses in the present experimental conditions, they were not analyzed. Several of the Thompson and Spencer (1966) criteria have, then, been fulfilled. A response decrement did occur with repeated stimulation. After as little as 1 hr, spontaneous recovery returned responsivity to initial levels. However, the rate of decrement after as much as 2 hr of spontaneous recovery was greater than that observed during acquisition, indicating potentiation of habituation. Dorsal nerve responsivity in surgical preparations recovers in as little as 12 min, and potentiation effects are not seen when intervals of 30 min separate series of stimuli (Lahue & Corning, 1971). A similar disparity between retention times in Aplysia preparations has also been reported (Carew, Pinsker, & Kandel, 1972). Responsiveness to a novel stimulus following habituation demonstrates that effector fatigue is not the basis for the response decrement, and sensory receptor fatigue had been ruled out in previous investigations of acute preparations (Lahue, 1974; Lahue & Corning, 1971).
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FIGURE 1. Percent change in telson muscle activity during the first 300 sec (11 trials) of an initial test and a retest after three different spontaneous recovery periods. (The initial test data for each of the 3 test-retest pairings have been combined.)
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EXPERIMENT 2 Having established that habituation occurs to the air puff and that some of the primary habituation criteria could be met, we examined further criteria in Experiment 2. The effect of the frequency of stimulation upon habituation and the influence of dishabituation stimuli upon responsivity to subsequent stimuli were investigated. Method A total of 11 animals was tested at four different stimulus frequencies. At least 48 hr intervened between successive tests on a single animal, and the order of presentation of the different frequency conditions was randomized. A total of 40 air-puff stimuli were presented with ITIs of 60 sec (n = 9), 30 sec (n = 9), 15 sec (n = 11), and 5 sec (n = 11). Between Trials 20 and 21 a jet of water (dishabituation stimulus) was directed at the stimulation site.
Results and Discussion The data presented in Figure 3 are plotted with respect to time on the abscissa.
Analyses of variance of the differences among the groups were performed on the data obtained 60 and 120 sec after the beginning of stimulation as well as immediately after the dishabituation stimulus, and 60 and 120 sec thereafter. Comparisons were also made between the 30- and 60-sec groups 540 sec after both the first stimulus and the dishabituation stimulus. Three analyses, then, were performed on the data obtained during the first 20 trials. After 60 sec of stimulation, there were significant differences between all groups (H = 24.53, p < .001; U < 24.5, p < .02 for six comparisons). After 120 sec, similar results were obtained (H = 20.77, p < .001; U < 24.5, p < .02 for six comparisons). At 540 sec, the 30- and 60-sec groups differed significantly (U = 69, p < .02). There were no differences among the groups in the magnitude of the response to the dishabituation stimulus (H = 3.97, p > .05). However, there were significant differences in responsivity to air puffs immediately following the
HABITUATION IN HORSESHOE CRAB
presentation of the jet of water (H = 21.15, p < .001; U < 22.0, p < .02 for six comparisons). There were no significant differences between the 5- and 15-sec groups 60 and 120 sec after the dishabituation stimulus. However, all other comparisons among the four groups were significant at these two points in time (H > 20.91; p < .001; U < 24.5, p < .02 for all comparisons). The 30- and 60-sec groups also differed from one another 540 sec after the dishabituation stimulus (U = 64, p < .004). Total response magnitudes (the sum of the responses at time intervals 0, 60, 120, and 540 sec) exhibited significant differences when comparisons were made with the 20 trials before the dishabituation stimulus and those after the dishabituation stimulus for each of the four groups (T < 6, p < .02 for all four comparisons). The amplitudes of the responses to the first air puff following the dishabituation stimulus were significantly larger than those elicited by the previous air-puff stimulus for all four groups (T < 5, p < .05 in each case). The usual relationship between frequency of stimulation and rate of habituation was obtained: Higher frequencies produced more rapid decrement. The rate of habituation at the different frequencies rather closely paralleled the decrement seen in dorsal nerve recordings, although less habituation was observed in the latter with a 60-sec ITI. The possibility of demonstrating response sensitization at higher frequencies of stimulation (e.g., 1-sec ITI), as was observed in dorsal nerve recordings (Lahue & Corning, 1973),
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was precluded by the duration of the telson response, which approached 5 sec. True dishabituation was demonstrated in that the response to air on the 21st trial exceeded that prior to the dishabituation stimulus. The recovery was not complete as the posthabituation response was not so large as that elicited by the first stimulation. The degree of recovery following the dishabituation stimulus and the subsequent rate of habituation were both related to the frequency of stimulation. EXPERIMENT 3 Two further habituation criteria were examined in this experiment. The effects of stimulus intensity upon habituation to airpuff stimuli were tested in the first part, and stimulus generalization to a spatial displacement of the stimulus site was investigated in the second part. Method A total of 11 animals was used in this experiment. All subjects were tested under high-stimulus intensity conditions; nine of these were also tested with a low-intensity stimulus, and the order of presentation of the high- and low-intensity conditions was randomized. At least 1 wk intervened between the two tests for a given animal. The intensities of the stimuli used in these experiments were controlled by altering the pressure in the air reservoir.2 The pressures used were either 1 Ib.2 (.07 kg/cm ; low intensity) or 8 Ib. (.56 kg/cm ; high intensity). A total of 20 air-puff stimuli was presented, with a 30-sec ITI followed by a dishabituation test with a jet of water. Nine of these animals were subsequently presented (a minimum of 6 days later) with two series of 20 air-
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puff .stimuli (30-seo ITI) at the normal intensity (4 lb.; .28 kg/em 2 ). After the initial 20 trials the stimulus was immediately moved to another site, approximately homo topic, on the contralateral side of the animal. An additional 20 stimuli were directed at this site. The responses quantified during each series were normalized separately as described above. The response on the first trial in each series was used as the 100% response for the calculation of subsequent trials in the same series. This was necessary to allow comparison between the Jwo series of responses because the initial responses elicited by stimulation of different sites often varied in amplitude.
Results and Discussion As seen in Figure 4, the total responsivity during high-intensity stimulation exceeds that during low-intensity stimulation (U = 8, p < .002). The high-intensity stimulus diminishes the rate of habituation. This is due, in part, to an initial period of incremental responding and suggests a dualprocess possibility (Groves & Thompson, 1970). State or sensitizing influences upon the output are thought to be enhanced by strong stimuli and to compete with the habituation process. Analysis of the response to the water jet (dishabituation stimulus) in the two groups shows that the high-intensity group is more responsive (U = 23, p < .05). This suggests that state has been activated and some residual influence upon responsivity is seen even after habituation. Previous experi-
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'»•*• .05). Although Thompson and Spencer (1966) considered this an important criterion for habituation, Hinde (1970) suggested that a certain amount of stimulus specificity is also characteristic of this process. Not all invertebrates demonstrate generalization, and some exhibit only one type (e.g., generalization only to stimuli of different modalities presented to the same site or only to stimuli of the same modality presented to different sites; see Corning & Lahue, 1972; Dyal & Corning, 1973). GENERAL DISCUSSION The data obtained in the present investigations demonstrate that most of the habituation criteria that are outlined by Thompson and Spencer (1966) can be met in a relatively intact, chronic Limulus preparation. With the previously obtained evidence that these criteria can be generally fulfilled in acute and surgically reduced preparations (Lahue, 1974), polythetic comparisons are possible with both intact and simplified preparations of other species. Although few investigators gathered information on all nine criteria, there are a sufficient number of examples to make some tentative phyletic comparisons. Thus, for example, in acute surgical preparations of the locust Schistocerca gregaria (Horn & Rowell, 1968; Rowell, 1971) most of the criteria were fulfilled (intensity effects were not tested). Very little spatial generalization of habituation was observed. The time course of spontaneous recovery was rather long compared with that of surgically limited Limulus preparations, al-
HABITUATION IN HORSESHOE CRAB
though similar to that observed in intact preparations. Krasne (1969, 1973; Krasne & Woodsmall, 1969) also demonstrated the same habituation criteria, including generalization but excluding dishabituation, in crayfish abdominal ganglia. Generally, differences in the time course of habituation are seen in intact versus acute preparations, and the process observed in acute preparations does not correspond fully with the properties of habituation shown in intact animals. The Thompson and Spencer criteria should not be considered as rigid "laws" around which all comparative statements concerning habituation persist or fall. They represent guidelines which will probably shift and expand as additional investigations explore more species with respect to multiple criteria. Some of the problems concerning these criteria have already been discussed (Hinde, 1970; Lahue, 1974), and it may be that certain aspects of the habituation process will become less critical; for example, in the present study there was no generalization of habituation when a homotopic site was stimulated. A specificity in the decremental process is thus obtained in the abdominal ganglia, but in Limulus visual-motor reflexes, cross-optic generalization had been observed (Coming & Von Burg, 1968). The generalization process may become less important as a criterion that is used to classify the general process of habituation amd more of a "subset marker" (Dyal & Corning, 1973). Those criteria that have greater phylogenetic ubiquity (decrement, spontaneous recovery, frequency effects) may be considered critical for the general category. Accordingly, in Limulus two types of habituation are possible: a decrement with stimulus generalization in visual-motor reflexes and one without generalization in tactile reflexes mediated by the abdominal ganglia. The usefulness of this criterion and others will be expanded when they prove sensitive to phyletic differences and to structural and physiological variations. To summarize, comparisons between acute and chronic studies show a number of similarities. The rates of habituation in both cases were similar over a range of stimulus frequencies. Although the higher frequencies
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of stimulation, which were used to reveal the dual process of habituation in prior studies (Lahue & Corning, 1973), could not be utilized, a higher stimulus intensity did produce the incremental phase. Responsiveness to novel stimuli after habituation was also similar in both cases. The reintroduction of normal stimulation after the novel stimulus resulted in higher responses for a few trials. An interaction of the dishabituation effect with stimulus frequency was found, although this has not been previously reported in the literature. Stimulus generalization was not demonstrable in the intact animal; previous research with acute preparations did not systematically examine whether generalization was obtainable in surgically reduced systems. Discovery of a dual process (increment and decrement) during repetitive stimulation of isolated ganglia added to the complexity of habituation in Limulus (Lahue & Corning, 1973). A dual-process theory, as originally put forth by Groves and Thompson (1970), seems likely to apply to a wide variety of invertebrate and vertebrate preparations. The evidence obtained with the present preparation indicates that in Limulus the two processes are observed in the intact animal and are not artifacts of surgical procedures. With more intense stimulation the incremental phase is apparent. The two-process theory is most likely incomplete. In acute preparations evidence has been obtained that a third process—sensory modulation— interacts with the incremental and decremental processes (Lahue & Corning, 1973). Since the overall characteristics of habituation are similar in both acute and intact cases, it can be hoped that in surgically reduced preparations the elucidation of processes and mechanisms will have relevance to intact animals. The striking difference between the two preparations was in the persistence or retention of the habituation effect. As with other simple-system preparations, the performance of the intact animal is superior (e.g., Krasne, 1973). One obvious possibility is that the surgery results in metabolic degeneration. It is also possible that in the acute preparation, the trauma of surgery in-
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R. LAHUE, L. KOKKINIDIS, AND W. CORNING
creases central state and thereby counteracts the acquisition and retention phases. A third possibility is that because of metabolic degeneration, deliberate surgical isolation, or undetermined damage, the system is reduced structurally, i.e., it is less complex. From direct examination of the relationship between system complexity and habituation, it is known that the more intact system performs best (Corning & Lahue, 1972; Lahuc & Corning, 1971). The failure to find significant and useful effects of stimulation on heart rate was unexpected, since prior studies had shown light and tactile effects (Corning & Von Burg, 1968; Von Burg & Corning, 1970). However, in these previous studies stimulation was longer and more intense and was not given repetitively with short ITIs for the purpose of examining habituation. The results of the present study suggest that with the particular stimulus characteristics that were used, the heart was not a good indicant of stimulation effects because habituation was very rapid and/or the stimuli were weak with respect to this system. The telson muscles, however, represent a sufficiently sensitive Finally, a question should be raised concerning the adaptive significance of habituation to tactile input. The locus of stimulation used in the present investigation was the surface of the gills. Since the animal is a bottom dweller and not prone to swim as an adult, normal movements will result in consistent stimulation of the gills. Additionally, normal respiratory activities of the gills result in tactile input as they fold over each other, an activity that produces regular stimulation. Both sources of input could conceivably be disruptive to normal biological and behavioral activities if habituation were not possible—the heart rate might be continually interrupted and the telson would be frequently jerking upward as the system responded to each burst of tactile input. REFERENCES Abraham, F., Palka, J., Peeke, H. V. S., & Willows, A. O. D. Model neural systems and strategies for the neurobiology of learning. Behavioral Biology, 1972, 7,1-24. Adolph, A. R. Recording of optic nerve spikes
underwater from freely-moving horseshoe crab. Vision Research, 1971,11, 979-983. Carew, T. J., Pinsker, H. M., & Kandel, E. R. Long-term habituation of a defensive withdrawal reflex in Aplysia. Science, 1972, 175, 451-454. Corning, W. C., Dyal, J. A., & Lahue, R. Intelligence: An invertebrate perspective. In B. Masterton, M. E. Bitterman, W. Hodos, & H. Jerison (Eds.), Evolution of the nervous system and behavior. Boston: Winston, in press. Corning, W. C., Dyal, J. A., & Willows, A. O. D. (Eds.)- Invertebrate learning (Vol. 1). New York: Plenum Press, 1973. (a) Corning, W. C., Dyal, J. A., & Willows, A. O. D. (Eds.). Invertebrate learning (Vol. 2). New York: Plenum Press, 1973. (b) Corning, W. C., Dyal, J. A., & Willows, A. O. D. (Eds.). Invertebrate learning (Vol. 3). New York: Plenum Press, 1974. Corning, W. C., Feinstein, D. A., & Haight, J. H. Arthropod preparation for behavioral, electrophysiological and biochemical studies. Science, 1965, 148, 394-395. Corning, W. C., & Lahue, R. Invertebrate strategies in comparative learning studies. American Zoologist, 1972, IS, 455-469. Corning, W. C., Lahue, R., & Von Burg, R. Supra esophageal ganglia influences on Limulus heart rhythm: Confirmatory evidence. Canadian Journal of Physiology and Pharmacology, 1971, 49, 387-393. Corning, W. C., & Von Burg, R. Behavioral and neurophysiological investigations of Limulus polyphemus. In J. Salanki (Ed.), Neurobiology of invertebrates. New York: Plenum Press, 1968. Dyal, J. A., & Corning, W. C. Invertebrate learning and behavioral taxonomies. In W. C. Corning, J. A. Dyal, & A. O. D. Willows (Eds.), Invertebrate learning (Vol. 1). New York: Plenum Press, 1973. Groves, P. M., & Thompson, R. F. Habituation: A dual-process theory. Psychological Review, 1970, 77, 419-450. Hinde, R. A. Behavioral habituation. In G. Horn & R. A. Hinde (Eds.), Short-term changes in neural activity and behavior. Cambridge, England: Cambridge University Press, 1970. Horn, G., & Rowell, C. H. F. Medium and longterm changes in the behavior of visual neurones in the tritocerebrum of locusts. Journal of Experimental Biology, 1968, 49, 143-169. Kandel, E. R., & Spencer, W. A. Cellular neurophysiological approaches in the study of learning. Physiological Reviews, 1968, 4$, 65-134. Krasne, F. B. Excitation and habituation of the crayfish escape reflex: The depolarizing response in lateral giant fibres of the isolated abdomen. Journal of Experimental Biology, 1969, BO, 29-46. Krasne, F. B. Learning in Crustacea. In W. C. Corning, J. A. Dyal, & A. O. D. Willows (Eds.), Invertebrate learning (Vol. 2). New York: Plenum Press, 1973.
HABITUATION IN HORSESHOE CRAB Krasne, F. B., & Woodsmall, K. S. Waning of the crayfish escape response as a result of repeated stimulation. Animal Behaviour, 1969,77,416-424. Lahue, R. Habitualion characteristics and mechanisms in the abdominal ganglia of Limulus polyphemus. Unpublished doctoral dissertation, University of Waterloo, 1974. Lahue, R., & Corning, W. C. Habituation in Limulus abdominal ganglia. Biological Bulletin, 1971, 140, 427-439. Lahue, R., & Corning, W. C. Incremental and decremental processes in Limulus ganglia: Stimulus frequency and. ganglion organization influences. Behavioral Biology, 1973,8,637-653. Lahue, R., & Corning, W. C. Synthesis: A comparative look at vertebrates. In W. C. Corning, J. A. Dyal, & A. O. D. Willows (Eds.), Invertebrate learning (Vol. 3). New York: Plenum Press, 1974. Rowell, C. H. F. Antennal cleaning, arousal and
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visual interneurone responsiveness in a locust. Journal of Experimental Biology, 1971, 55, 749761. Siegel, S. Nonparametric statistics. New York: McGraw-Hill, 1956. Sneath, P., & Sokal, R. Principles of numerical taxonomy. San Francisco: Freeman, 1973. Thompson, R. P., & Spencer, W. A. Habituation: A model phenomenon for the study of the neuronal substrates of behavior. Psychological Review, 1966, 178, 16-43. Von Burg, R., & Corning, W. C. Cardioregulatory properties of Limulus abdominal ganglia. Canadian Journal of Physiology and Pharmacology, 1970, 48, 333-341. Von Burg, R., & Corning, W. C. The effects of drugs on Limulus cardioregulators. Canadian Journal of Physiology and Pharmacology, 1971, 49, 1044-1048. (Received November 21, 1974)
